Fructose-2,6-bisphosphate (Fru-2,6-P2) modulates the rate of glycolysis via its potent activation of phosphofructokinase (PFK). The intracellular concentration of Fru-2,6-P2 is determined by the balance of the antagonistic kinase and phosphatase activitie of the unique bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6-PF-2-K/Fru-2,6-P2 ase) The need for glycolytic activity varies both with time and from tissue to tissue. Thus tissue specific isoforms of 6-PF-2-K/Fru-2,6-P2 ase have been identified for several cell types (liver, skeletal muscle, heart muscle, testis, placenta, and brain). Each of these isoforms has a characteristic relative ratio of kinase to phosphatase activity, and are uniquely regulated b various inhibitors and activators which vary in concentration with the metabolic needs of the cell (i.e., PEP, Pi NTPs, citrate, phosphoglycerate). The activities of the liver and heart isozymes are further regulated by hormon induced protein phosphorylation of NH2- and COOH-terminal regulatory domains respectively. The phosphorylation of the liver isozyme is stimulated by glucagon, and results in inhibition of the 6-PF-2-K activity, with a concomitant activation of the Fru-2,6-P2 ase. The phosphorylation of the heart isozyme is stimulated by epinephrine, and results in an activation of the 6-PF-2-K activity, with no effect on the Fru-2,6-P2 ase. So not only are the sites of phosphorylation for these two isozymes at opposite ends of the polypeptide chain, but they have opposite effects on the 6-PF-2-K activity as well. This system offers a unique opportunity to observe how a single enzyme has evolved to balance two antagonistic catalytic activities in a tissue specific fashion, involving both ligand-mediated and phosphorylation-dependent mechanisms for the regulation of catalysis. It is the goal of the proposed research to determine the molecular basis of these phenomena. They have alread determined the 2.0Aring; structure of the unregulated rat testis isozyme. They will now determine the three dimensional structure of the testis isozyme in various ligand-bound conformations. In addition, they will crystallize and solve the structure of the liver, heart, and placenta isozymes, in both their phosphorylated and unphosphorylated states.